The invention is in the general field of apparatus for containing and conducting chemical reactions. The invention also provides methods of using the apparatus to provide control over one or more chemical reactions in a series.
Chemical reactions encompass molecular interactions such as the formation and cleavage of covalent bonds and ionic bonds; the association or dissociation of two or more chemical compounds; and changes in primary, secondary, tertiary, or quaternary structure. Chemical reactions include nonenzymatic and enzymatic reactions. Whether or not enzymes are present, a chemical reaction is usually made of several mechanistic steps or molecular interactions, including conformational changes, transition state formation, electron or proton donation/acceptance, and electron rearrangement. Typically, a series of chemical reactions provides a useful chemical product.
For example, in molecular biology, nested sets of deletions are used to create site-directed mutants which are used to probe the function of DNA segments in both structural and regulatory gene sequences. A collection of nested deletions within a gene allows the fine mapping of regions such as enhancers, promoters, and termination sites which are necessary for regulatory functions; and, regions having structural function, such as those defining domains within proteins. It is desirable to create deletions which vary only slightly from each other, for example, by 10-20 base pairs. Existing methods for generating nested sets of deletions digest double stranded DNA with nucleases including restriction endonucleases, Bal31, pancreatic DNase I (DNase I), and Exonuclease III (Exo III).
Restriction endonucleases are used to partially digest a DNA template which contains multiple sites for a given restriction endonuclease. This method requires prior knowledge of restriction sites within a DNA template. Because restriction sites are not randomly distributed throughout a DNA template, many DNA templates will not contain sufficient or properly spaced restriction enzyme sites to generate a useful set of deletion mutants. This is particularly problematic when the mutants are intended to delineate the boundary of regulatory domains.
Turning to another endonuclease, Bal 31 digests double stranded linear DNA from both the 5xe2x80x2 and 3xe2x80x2 termini. To create a set of unidirectional mutants, a double stranded DNA template (plasmid, phage, or replicative form of M13) is linearized with a restriction enzyme which cleaves at one end of the target sequence. The linearized DNA is incubated with Bal 31. Varying time and the amount of enzyme respectively control the extent of digestion and the rate of digestion. Most commercial preparations of Bal 31 contain two distinct forms of the enzyme, a fast and a slow form, the latter being a proteolytic fragment of the former. The extent of digestion depends on the proportion of the two forms.
Each batch of Bal 31 is therefore assayed to determine suitable digestion conditions.
Bal 31 is a processive enzyme which simultaneously degrades both the target DNA and the flanking vector DNA. Bal 31 activity varies with the primary structure of the DNA template; A-T rich,regions are degraded faster than G-C rich regions. Recovery of the truncated target fragments and subcloning into an appropriate vector is required. The processive properties result in the generation of heterogeneous deletions. Bal 31 requires purification because it is inhibited by the presence of RNA.
Turning to a third enzyme, pancreatic DNase I will cut double stranded DNA templates at about the same location on both strands, in the presence of transition metal ions such as Mn2+ or Co2+. Incubation of closed circular DNA with DNase I generates a set of linear molecules which are cut at locations randomly dispersed throughout the target DNA. A portion of the starting material is never converted to the linear form. After a restriction enzyme cleaves at one end of the target sequence, the sequences are repaired using DNA polymerase, and recircularized. The fraction of clones recovered using this technique can be quite small. DNase I can generate deletions in target DNA contained within, for example, plasmid, phage, or the replicative form of M13 vectors [G. F. Hong, J. Mol. Biol. 158:539 (1982) and Methods Enzymol. 155:93 (1987); S. Labeit et al., Methods Enzymol. 155:166 (1987)].
One method of generating nested sets of deletions is digestion of double stranded DNA with Exo III [L.-H. Guo and R. Wu, Methods Enzymol. 100:60 (1983); S. Henikoff, Gene 23:351 (1984) and Methods Enzymol. 155:156 (1987)]. Exo III degrades double stranded DNA molecules in a 3xe2x80x2 to 5xe2x80x2 direction from either a 5xe2x80x2 overhang or a blunt end. Nested deletions are, generated by digesting the double stranded DNA with two restriction enzymes whose cleavage sites lie, between one end of the target and the binding site for the universal sequencing, primer on the vector. The restriction enzyme cutting nearest to the target DNA must generate either a blunt-end or a 5xe2x80x2 overhang. The other enzyme must generate a 3xe2x80x2 overhang. As Exo III cannot degrade DNA having a 3xe2x80x2 overhang, digestion of the doubly restricted molecule proceeds in a unidirectional manner. Following Exo III digestion for varying lengths of time, the single-stranded regions are removed with a single-strand nuclease such as Mung Bean nuclease. The DNA is then repaired and recircularized. The extent of Exo III digestion is controlled by varying the length of the incubation period. In addition, the temperature may be lowered to decrease the rate of digestion [G. Murphy, in DNA sequencing Protocols, H. G. Griffin and A. M. Griffin, eds., Humana Press (1993) p. 58]. This method requires two restriction enzymes which satisfy the above conditions and which do not cut within the target DNA. This requirement is difficult to satisfy when the target DNA is long.
The invention features methods and apparatus in which pressure provides precise control over the timing and preferably synchronization of chemical reactions, particularly enzymatic reactions. The disclosed apparatus enables automated and generally rapid changes in pressure. In turn, these pressure changes control chemical reactions, and can control single, pressure-sensitive chemical events, such as the cleavage or addition of a single amino acid or nucleotide. Control and detection of chemical events is particularly useful for synthesizing and characterizing heteropolymers such as nucleic acids and polypeptides.
One aspect of the invention features a pressure cycling reactor which produces programmable fluctuations in the reaction vessel pressure. Preferably the pressure cycling reactor is capable of rapid programmable fluctuations, such as net changes in vessel pressure of about 10,000 to 30,000 psi or more which can be achieved in hundreds of milliseconds or less. The transition time between one pressure and another pressure can be 250 milliseconds, 150 milliseconds, 100 milliseconds, 50 milliseconds, or 30 milliseconds or less.
Pressure is controlled during a sequence of changes. For example, a first pressure P1 is a reaction inhibitory pressure which can be changed to a second pressure P2, a reaction permissive or enabling pressure. The permissive pressure is maintained for a controlled period of time. Then the pressure is changed to a third pressure P3, a reaction inhibitory pressure. Some embodiments permit the addition and removal of reaction mixture components while maintaining the reaction mixture pressure, whether at P1, P2, or P3.
A pressure pulse or pressure cycle is the event including (i) a change from a first to a second pressure, (ii) maintenance of the second pressure for a period of time, and (iii) a change from the second pressure to a third pressure. The first pressure and the third pressure may be substantially different, or may be substantially the same. The second pressure is a pressure sufficient to affect the contents of the reaction vessel in the desired manner, usually enabling a reaction or reaction step to occur.
According to the invention, the pressure cycling reactor includes a reaction vessel for containing a sample, a vessel pressurizer connected to the reaction vessel, and a controller for signalling the pressurizer to maintain reaction inactivating pressure in the reaction vessel and signalling the pressurizer to change pressure in the reaction vessel to a reaction activating pressure for a predetermined short pulsed period and then recycling to a reaction inactivating pressure in the reaction vessel.
In particular embodiments of the invention, the pressurizer changes pressure by over 20,000 psi in less than about 250 milliseconds. The vessel pressurizer includes a pressure chamber, e.g., a pneumatic pump cylinder, connected to the reaction vessel such that the pressure chamber communicates with the reaction vessel responsive to the controller, and a pressure transmitter, e.g., a pneumatic cylinder, connected to a pressure source. The pressure chamber has a variable volume and the position of a pressure chamber wall is controlled by the pressure transmitter to control the volume of the pressure chamber.
A relief valve is used to quickly adjust the pressure in the pressure transmitter thereby creating a pressure pulse in the reaction vessel. A pressure chamber outlet valve is positioned between the pressure chamber and the reaction vessel. A pressure chamber inlet valve is positioned between a fluid source which communicates with the reaction vessel and the pressure chamber. The fluid source includes a fluid reservoir for connection to a reservoir pressure source, and a reservoir control valve, whereby fluids are moved from the fluid reservoir to the reaction vessel via the pressure chamber, while pressure in the reaction vessel is controlled. The temperature of the fluid in the reservoir is controlled by a temperature sensor connected to the fluid reservoir and a reservoir vessel heating and/or cooling source. The temperature of the reaction vessel is controlled by a temperature sensor connected to the reaction vessel and a reaction vessel heating and/or cooling source. The controller monitors the pressure in the reaction vessel with a pressure sensor connected to the reaction vessel. A pressure regulator adjusts the pressure in the pressure chamber and the controller uses feedback from the pressure sensor to control the pressure regulator.
A second vessel pressurizer is connected to the reaction vessel whereby fluids are removed from the reaction vessel while pressure in the reaction vessel is controlled. An inlet valve is located between the reaction vessel and the second vessel pressurizer. An outlet valve is located between the second vessel pressurizer and a second fluid reservoir. A second reservoir control valve is connected to the second fluid reservoir. The controller controls a plurality of valves according to a predetermined set of stored signals which control the valves to add at least one pressurized reagent fluid to the reaction vessel and to remove at least one pressurized reacted fluid from the reaction vessel, while the reaction vessel remains under pressure. A detector, e.g., a radioisotopic detector, an infra-red spectrometer, a mass spectrometer, a gas chromatography-mass spectrometer, a spectrophotometer, a spectrofluorometer, an electrochemical detector, a surface plasmon resonance detector, or a photometer, detects a characteristic of a component present in fluid in or removed from the reaction vessel.
The reaction vessel includes a restraint, e.g., a semi-permeable barrier which divides the reaction vessel into two segments to retain immobilized reagent, e.g., an organic compound attached to a non-liquid support, within the reaction vessel while permitting removal of fluid from the vessel.
According to another aspect of the invention, a reactor for intermittently inhibiting activity of a sample by controlling temperature and pressure conditions of the sample includes a pressurizer in communication with a sample chamber and mounted for movement between a first position for applying a first predetermined pressure to the sample chamber selected to inhibit activity of the sample and a second position for changing the chamber pressure to a second predetermined pressure selected to allow activity of the sample; a switch having a first state and a second state to move the pressurizer between the first position and,the second position; a controller for changing the switch between the first and second states; means for adjusting temperature in the chamber; and a port in communication with the chamber for removal of the sample from the chamber.
According to another aspect of the invention, a reactor for intermittently inhibiting activity of a sample by controlling pressure applied to the sample includes a system for providing flow of test reagent from a fluid reservoir into a sample chamber while the chamber is pressurized. The system includes a first valve located in a first conduit in communication with the fluid reservoir; a second valve located in a second conduit in communication with the sample chamber, a first pressurizer located between the fluid reservoir and the chamber and in communication with the first conduit and the second conduit; and a third valve associated with the first pressurizer for venting the first pressurizer.
With the first valve in an open position and the second valve in a closed position, the reservoir is in communication with the first pressurizer to allow fluid flow to the first pressurizer; with the first valve in a closed position, the second valve in an open position, and the third valve in a closed position, the pressurizer is in communication with the chamber to pressurize the chamber; with the first valve in a closed position, the second valve in an open position, and the third valve cycled between an open position and a closed position, the pressure in the chamber is pulsed.
In particular embodiments of this aspect of the invention, to provide for flow of the test material out of the chamber, the system includes a fourth valve located in a third conduit in communication with the chamber; a fifth valve located in a fourth conduit downstream of the third conduit; a second pressurizer located downstream of the chamber and in communication with the third conduit and the fourth conduit, and a sixth valve associated with the second pressurizer for venting the second pressurizer.
With the first valve in a closed position, the second valve in an open position, the third valve in a closed position, and the fourth valve in a closed position, the pressurizer is in communication with the chamber to pressurize the chamber; with the first valve in a closed position, the second valve in an open position, the third valve cycled between an open position and a closed position, and the fourth valve in a closed position the pressure in the chamber is pulsed; and with the first valve in a closed position, the second valve in an open position, the third valve in a closed position, the fourth valve in an open position, the fifth valve in a closed position, and the sixth valve in an open position the first pressurizer is in communication with the second pressurizer to enable flow through from the first pressurizer through the chamber to the second pressurizer.
One aspect of the invention features a method of controlling an enzymatic reaction step with a rapid pressure cycle or pressure pulse. This method includes providing a sample mixture in a sample vessel at a pressure Pi,x at which an enzymatic reaction step is reversibly inhibited (x is an integer xe2x89xa70). The sample mixture includes an enzyme. The method also includes the step of changing the pressure of the sample mixture in a length of time xcex4ta,y to pressure Pa,y at which the enzymatic reaction step can occur (y is an integer xe2x89xa7 to 1). The method also includes the step of changing the pressure of the sample mixture in a length of time xcex4ti,zto pressure Pi,z at which an additional enzymatic reaction step is reversibly inhibited, where z is an integer greater than or equal to 1, thereby controlling the enzymatic reaction step.
In addition to an enzyme, the sample mixture can include one or more of each of the following in various combinations: a solvent, an enzymatic cofactor, a substrate of the enzyme, an enzymatic inhibitor, a substrate mimetic, and inorganic or organic ions. In most embodiments, the sample mixture includes a substrate. The sample mixture can also include materials on which components of the sample mixture are immobilized for post-enzymatic reaction step 5 retrieval, by which components of the sample mixture are retained within a space of the sample vessel; or by which products or byproducts of an enzymatic reaction are scavenged, adsorbed, associated, or bound by covalent or noncovalent interaction.
One embodiment therefore generates nested deletions wherein the extent of digestion can be finely controlled. Varying amount of enzyme, length of incubation and temperature at atmospheric pressures in addition to pressure results in finer control of the extent of digestion, than methods wherein the pressure is about atmospheric pressure. This embodiment provides groups of deletions having widely or tightly clustered lengths.
The invention features methods of controlling an enzymatic reaction. These methods include providing a sample mixture in a sample vessel at reversibly inactivating pressure, the sample mixture containing an enzyme; exposing the sample mixture to activating pressure; and (iii) exposing the sample mixture to inactivating pressure, thereby controlling an enzymatic reaction. The components of the sample mixture (e.g., one or more substrates of the enzyme, cofactors, transition metal ions, solvent, salts, and buffers) may be provided in any order appropriate to the particular enzymatic reaction or reaction step to be controlled. The enzyme can have distributive or processive properties.
The inactivating pressure is Pi,x in step (i), at which pressure an enzymatic reaction step is reversibly inhibited; the activating pressure is Pa,y in step (ii) at which pressure the enzymatic reaction step can occur, the exposing step (ii) comprising changing the pressure to Pa,y in a length of time xcex4ta,y; and the inactivating pressure is Pi,z in step (iii), at which an additional enzymatic reaction step is reversibly inhibited, the exposing step (iii) comprising changing the pressure to Pi,z in a length of time xcex4ti,z; x being an integer greater than or equal to zero, y being an integer greater than or equal to 1, and z being an integer greater than or equal to 1, thereby controlling the enzymatic reaction.
The method can further include the following steps:
between steps (ii) and step (iii), the activating pressure Pa,y is maintained for a time period ta,y corresponding to the average length of a single enzymatic event; or where the substrate is immobilized within the sample vessel, after step (iii) the step of removing a component of the sample mixture from the sample vessel while maintaining the sample vessel pressure, or the step of adding a liquid to the sample mixture while maintaining the sample vessel pressure.
Removed components include a restriction endonuclease, a restriction endonuclease cleavage product, a exonuclease, a nucleotide, or a combination thereof. The removed component can pass through a semi-permeable material when the component is removed from the sample vessel.
The method can further include after step (iii) the step of detecting a characteristic of a component of the sample mixture. Component characteristics include radioactivity, fluorescence, chemiluminescence, molecular ion charge/mass ratio, electrochemical potential, light emission, surface plasmon resonance, and infra-red absorption.
The substrate can be a nucleic acid (e.g., double stranded DNA, single stranded DNA, DNA containing both double and single stranded regions, and RNA, wherein any of the foregoing is immobilized or not immobilized within the sample vessel; and combinations thereof). The enzyme can be a restriction endonuclease, an exonuclease, or a terminal transferase.
The methods can therefore result in at least one cleavage fragment (e.g., nucleotide, amino acid, or oligomer) being cleaved from the nucleic acid substrate.
In one method, there are a first substrate and a second substrate, wherein the enzyme acts to attach the first substrate to the second substrate. In one embodiment, the first substrate is a nucleotide; the second substrate includes an RNA oligonucleotide or a DNA nucleotide; and the enzyme is a polyribonucleotide phosphorylase where the second substrate is an RNA oligonucleotide and a terminal transferase where the second substrate is a DNA nucleotide.
In another embodiment, the first substrate is a nucleotide; the second substrate comprises an RNA or DNA oligonucleotide; and the enzyme is a transferase selected from the enzyme class 2.7.7.
In one embodiment, the substrate is a compound with a chiral or pro-chiral functional group; and the enzyme is a protease, a dehydrogenase, an oxidase, a transferase, a lipase, or an esterase acts on the substrate enantio-specifically.
In another aspect, the enzyme in steps,(i)-(iii) is a first enzyme, and the sample mixture in steps (i)-(iii) is a first sample mixture. This aspect further includes after step (iii) the following steps (iv)-(vi): (iv) providing a second sample mixture in a sample vessel, the sample mixture comprising a second enzyme at a reversibly inactivating pressure Pi,j, the second enzyme being the same as or different from the first enzyme in steps (i)-(iii), and the sample vessel being the same as the sample vessel in steps (i)-(iii) or being a second sample vessel connected to the first sample vessel by a valve; (v) exposing the second sample mixture to a reversibly inactivating pressure; and (vi) exposing the second sample mixture to a reversibly activating pressure, thereby controlling enzymatic reaction steps of the first and second enzymes.
The transition time to pressure Pa,k occurs in a time period xcex4ta,k to pressure Pa,k at which the enzymatic reaction step of the second enzyme can occur. The first and second enzymes can be the same enzyme or different enzymes. The pressure of the second sample mixture is changed in a time period xcex4ti,l to pressure Pi,l at which an additional enzymatic reaction step of the second enzyme is reversibly inhibited. In some embodiments, reaction inactivating pressure Pi,z in step (vi) is substantially the same as Pi,l.
According to one embodiment, the sample mixture at pressure Pi,x is at temperature Ti,x whereby the enzyme is inhibited; the sample mixture at pressure Pa,y is at temperature Ta,y, whereby the enzyme is active; and the sample mixture at pressure Pi,z is at temperature Ti,Z whereby the enzyme is inhibited; each of Ti,z and Ta,y being independently the same as or different from Ti,x. The values for j, k, and l are the same as x, y, and z, respectively. These subscripts are intended to emphasize that additional steps in pressures may be included, between the described steps (e.g., between step (i) and (ii), there may be a change to a different, not necessarily intermediate, pressure. The method steps can be combined as either repetitive cycles or as nonrepetitive changes in pressure and time, depending on the particular product desired. Moreover, the transition time periods xcex4ts are preferably short, e.g., less than 1 second, preferably less than 500, 250, 200, 100 or 50 milliseconds or each of xcex4ta,y and xcex4ti,z is between 10 and 250 milliseconds or 10 and 50 milliseconds. A pressure may be maintained for a time period, e.g., ta,y or ti,z that range from spike-like transient changes without any measurable plateau to hundreds of milliseconds to seconds (see FIG. 11). For example, the sum (xcex4ta,y+ta,y+xcex4ti,z) is less than or equal to 300, 700, or 1000 milliseconds.
Embodiments also include methods wherein the substrate is immobilized within the sample vessel and the second enzyme is different from the first enzyme, further including between steps (iii) and (iv) the step of removing the first enzyme from the sample vessel while maintaining the sample mixture pressure by eluting with an eluting solution; and methods in which the substrate is a double stranded nucleic acid, the first enzyme is a 5xe2x80x2-3xe2x80x2 exonuclease, and the second enzyme is a 3xe2x80x2-5xe2x80x2 exonuclease, thereby identifying one or more nucleotides by sequencing with the first enzyme and confirming the one or more nucleotides by sequencing with the second enzyme.
Where steps (i)-(iii) are one cycle, a method can include the steps of repeating the cycle of steps (i)-(iii) at least 49 times, wherein the value for each respective value of Pi,x, Pi,z, xcex4ta,y, Pa,y, and xcex4ti,z in a cycle, is independent of the respective value in any other cycle.
Where steps (iv)-(vi) are one cycle, a method can include the steps of repeating the cycle of steps (iv)-(vi) at least 49 times. A fluid can be added to (or a component removed from) the sample mixture while maintaining the pressure of the sample mixture;
Another aspect is a method of affecting the thermodynamic equilibrium of a reaction, including (i) providing a sample mixture in a sample vessel, the sample mixture being at a pressure P0 and a temperature T0; (ii) changing the sample mixture temperature to T1; (iii) increasing the pressure of the reaction mixture to P1 in a length of time xcex4t1, wherein P1 is at least 10,000 psi greater than P0; and (iv) reducing the pressure of the reaction mixture to P2, thereby affecting the thermodynamic equilibrium of the reaction at high pressure.
Embodiments of this aspect include, after the pressure increasing step (iii) the further step of allowing the sample mixture to react; or in which the sample mixture includes a catalyst, after the pressure increasing step (iii) the further step of allowing the product to dissociate from the catalyst; and after step (iii) the further step of removing a component of the sample mixture from the sample vessel.
Another aspect provides a method for treating nucleic acid, including a) providing, in any order: i) a sample vessel, ii) a nucleic acid substrate, iii) an enzyme capable of acting on the nucleic acid substrate, and iv) a pressurizer controlling pressure in the vessel; b) providing the enzyme and tile nucleic acid in solution in the sample vessel and maintaining the enzyme under inactivating pressure conditions; and c) changing the pressure in the sample vessel to an enzyme activating pressure for a controlled period of time, such that the enzyme is active and acts on the nucleic acid substrate for the time period.
The enzyme can be an exonuclease (e.g., Lambda exonuclease), a DNA polymerase or a RNA polymerase; a processive enzyme; a distributive enzyme. The enzyme can modify the nucleic acid substrate, after which a reaction product, such as a cleaved nucleotide, amino acid, or modified substrate, is detected.
The method also includes controlling reaction vessel temperature, wherein the inactivating pressure conditions include a temperature that permits a high level of enzyme activity when reaction vessel pressure is reduced to the enzyme activating pressure. The method can include maintaining the reaction vessel at a low, enzyme inactivating temperature (e.g., less than approximately 5xc2x0 C.) and an enzyme activating pressure (e.g., approximately 5,000 to 15,000 pounds per square inch), raising the pressure to an enzyme inactivating pressure, lowering the temperature to an enzyme activating temperature (e.g., an integral temperature between 15xc2x0 C. to 20xc2x0 C.), and then lowering the pressure to an enzyme activating pressure.
For example, the method includes a) maintaining the enzyme at an inactivating temperature of less than approximately 5xc2x0 C., thereby rendering the enzyme substantially inactive; b) adding the nucleic acid substrate to the inactive enzyme to create a reaction mixture; c) increasing the pressure in the sample vessel to an enzyme inactivating pressure of greater than approximately 30,000 pounds per square inch; d) raising the temperature of the reaction mixture in the sample vessel to greater than approximately 10xc2x0 C.; and e) for a controlled period of time, lowering the pressure in the sample vessel to an enzyme activating pressure of less than approximately 20,000 pounds per square inch, thereby rendering the enzyme active such that the enzyme acts on the nucleic acid substrate. The method can include f) raising the pressure in the sample vessel to an enzyme inactivating pressure.
The method can also include the further steps of steps of changing the pressure in the sample vessel to an enzyme inactivating pressure; and repeating a cycle of steps c) and d) at least once, at least five times. The enzyme inactivating pressure is generally higher than the enzyme activating pressure.
Other features and advantages of the invention will be apparent from the following description of the drawings, the detailed description, examples, and from the claims.